blmp-1在線蟲遠頂細胞遷移中的異時性調控

Abstract

在線蟲(Caenorhabditis elegans)的雌雄同體中，兩顆distal tip cell(DTC)會在幼蟲期進行三個時期不同方向的遷移。之前的研究發現，netrin guidance system會影響第二時期的遷移，在野生型線蟲中，netrin的受器UNC-5的表現就足以使DTC進行第二時期的遷移。另外，有三個異時基因dre-1、daf-12與lin-29也被發現會共同促進DTC第二時期的遷移。我們實驗室之前發現了一個新基因blmp-1也會影響第二時期的遷移。當blmp-1突變時，DTC會比在野生型線蟲中提早第二時期遷移。根據遺傳分析，我們認為blmp-1應在dre-1、daf-12的下游，與lin-29位於不同路徑或在其下游，並在unc-5的上游。此外，我們亦發現在dre-1、daf-12與lin-29其中兩個基因突變下，BLMP-1會持續存在，而且BLMP-1的過量表現會抑制unc-5的表現。這些資料顯示blmp-1需被dre-1、daf-12與lin-29三個異時基因向下調控，使DTC得以進行第二時期的遷移。由於此三個異時基因所轉譯出的蛋白質具有不同的功能，DRE-1是F-box protein，DAF-12是荷爾蒙受器，LIN-29是鋅指轉錄因子，我們推測blmp-1可能會在轉錄、轉錄後及轉譯後幾個階層受到向下調控。為了確認blmp-1是在何階層受到向下調控，我們將blmp-1分為三個部份結合螢光蛋白追蹤分析，分別是啟動子、3端不轉譯區以及cDNA。我們發現lin-29會在第二時期遷移後抑制blmp-1的轉錄，dre-1與daf-12則是會在mid L3降解BLMP-1。blmp-1的3端不轉譯區在blmp-1的向下調控中則是不具有功能。由此可知blmp-1會在不同時間點受到不同層次上的調控，而BLMP-1的降解，可能是DTC遷移中，由第一時期調控網絡轉移成第二時期調控網絡的重要因子。

In the hermaphrodite of Caenorhabditis elegans, two distal tip cells (DTCs) undergo three-phased migration during larval stages. Previous studies have shown that the netrin guidance system functions in phase II migration, and the expression of UNC-5, a receptor of netrin, is sufficient for promoting phase II migration in wild type. Three heterochronic genes, dre-1, daf-12, and lin-29, which encode F-box protein, nuclear hormone receptor, and zinc finger transcription factor respectively, have been identified to function redundantly in promoting phase II migration. A previous study in the lab has identified a novel heterochronic gene blmp-1 also regulating phase II migration. When blmp-1 is mutated, DTCs initiate phase II migration earlier at early L3 rather than late L3. In addition, genetic analyses suggest that blmp-1 acts downstream or parallel of dre-1, daf-12 and lin-29, and is down-regulated for promoting phase II migration. Therefore, my thesis focuses on dissecting the mechanisms by which dre-1, daf-12, and lin-29 regulate the expression level of blmp-1. To verify at which level blmp-1 is down-regulated, I separated the blmp-1 gene into three regions, promoter, 3’UTR, and cDNA, and then fused them with different reporter genes and assessed their expression during DTC migration. We find that reporter genes fused with promoter or cDNA region of blmp-1 show down-regulation during L4 and at mid L3 respectively, but the 3’UTR region not. Furthermore, we find that the down-regulation of reporter genes fused with promoter region of blmp-1 is abolished by lin-29 RNAi treatment, while down-regulation by cDNA region of blmp-1 is eliminated in dre-1 or daf-12 mutant. These data suggest that lin-29 suppresses the transcription of blmp-1 after phase II migration, while dre-1 and daf-12 degrade BLMP-1 at mid L3. These results imply that blmp-1 is down-regulated at different levels and times and the protein degradation might be the key point for the transition of network regulating DTC migration from phase I to phase II.